DESCRIPTION (from the application):The long-term goals of this proposal are to understand the detailed mechanism by which splicing enhancers function in humans and other metazoans, and to identify new human splicing regulatory proteins. In many cases, alternative splicing in metazoans is regulated by RNA sequence elements called splicing enhancers, which are typically located within exons downstream of the intron they regulate. Splicing enhancers are recognized by members of' the SR protein family of essential splicing factors. SR proteins contain an N-terminal RNA binding domain and a C-terminal arginine/serine-rich (RS) domain that functions as a splicing activation domain. The mechanism by which enhancer-bound SR proteins function to activate splicing is currently unclear. One model proposes that SR proteins recruit the splicing factor U2AF to the pre-mRNA while other models propose that U2AF binding is unaffected by splicing enhancers. Biochemical methods and an in vivo protein-RNA cross-linking technique will be used to discriminate between these two models for splicing enhancer function I experiments are also described in which the protein sequences that are required for splicing activation domain function are determined. The role of phosphorylation on the function of SR proteins in enhancer-dependent splicing will also be addressed. Moreover, the structure of a splicing activation domain will be determined by a combination of NMR and X-ray crystallographic approaches. Finally, new splicing regulators will be identified in a screen for proteins that can function to activate splicing in mammalian cells. At least 15 percent of all human diseases are the result of mistakes made by the splicing machinery in selecting the correct splice sites. Alternative splicing requires the splicing machinery to choose between the use of alternative splice Sites. Thus understanding how splice sites are selected in alternative splicing is of direct relevance to human health. In addition, at least 35 percent of human genes are alternatively spliced. Our results will increase our understanding of how this important means of gene regulation is controlled.